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- 16 Ivana D. Stevanović et al.
elevated levels of NO that are apparently involved in experiments. Animals were housed 2 or 3 per cage (Erath,
neurodegeneration by different mechanisms, including Germany) in an air-conditioned room at a temperature of
o
oxidative stress and activation of intracellular signaling 23 ± 2 C, 55 ± 10% humidity and with lights on 12 h/d
mechanisms [19]. (07:00-19:00). The animals were given a commercial rat
Al affects NO production in the brain. Al decreases diet and tap water ad libitum. Animals used for these
glutamine synthase activity and also increases extracellular procedures were treated in strict accordance with the NIH
glutamate, which leads to developing the excitotoxic process Guide for Care and Use of Laboratory Animals (USA).
that is under the influence of NO. Extracellular glutamate
increases the influx of Ca, NOS activity and, consequently, Chemicals
increased NO synthesis [5]. All chemicals were of analytical grade or better. AlCl3 was
Molecular oxygen (O2) is the primary biological electron purchased from Sigma (USA); L-NAME was purchased
acceptor that plays vital roles in fundamental cellular functions. from Sigma (USA); saline solution (0.9% w/v) was provided
However, concomitant with the beneficial properties of O2 by the Hospital Pharmacy (Military Medical Academy,
comes the inadvertent formation of reactive oxygen species Serbia). All drug solutions were prepared on the day of the
-
(ROS), such as superoxide anion radical (∙O2 ), hydrogen experiments.
peroxide (H2O2) and hydroxyl radical (∙HO) [30].
Accumulating evidence shows that iron (Fe) accumulates in Experimental procedures
-
the brain and catalyzes ∙O2 formation, which reacts with The rats were anesthetized by intraperitoneal injection of
-
NO to form the very toxic peroxynitrite anion (ONOO ) [20]. pentobarbital sodium (45 mg/kg b.w.) before
Free radicals (oxidative toxins) have been implicated in the intrahippocampal administration of the following: (1)
control group (n = 8) treated with 10 μl of 0.9% saline
destruction of cells via lipid peroxidative damage to cell
membranes. After exposure to Al, increased malondialdehyde solution; (2) AlCl3 group (n = 15) - animals were treated
-4
(MDA), an index of lipid peroxidation, was observed [35]. with AlCl3 with 1 single dose (3.7 × 10 g/kg b.w. in 0.01
In normal cells, oxygen derivatives are neutralized or ml of deionizied water); (3) L-NAME + AlCl3 group (n =
eliminated by a natural defense mechanism that involves 10) - animals were pre-treated with L-NAME with 1 single
-4
enzymatic anti-oxidants (glutathione peroxidase, superoxide dose (1 × 10 g dissolved in saline) before AlCl3
dismutase, catalase) and water or fat-soluble non-enzymatic administration; (4) L-NAME group (n = 10) - animals were
-4
anti-oxidants (vitamins C and E, glutathione, selenium) [32]. treated with L-NAME with 1 single dose (1 × 10 g dissolved
The antioxidant thiol L-gamma-Glutamyl-L-cysteinyl- in saline) before saline administration.
glycine, or glutathione (GSH), has shaped, and is still refining, Using a stereotaxic instrument for small animals, chemicals
the nature of oxidative signaling in terms of regulating the were injected by Hamilton microsyringe into the CA1
milieu of inflammatory mediators, ostensibly via the sector of the hippocampus (coordinates: 2.5 A; 4.2 L; 2.4
modulation of oxygen- and redox-responsive transcription V) [16]. L-NAME was immediately injected before the
factors. Hence, they are termed redox(y)-sensitive cofactors neurotoxin/saline solution. For all treated animals, the
injected intracerebral volume was 10 μl and was always
[12]. Also, anti-oxidative defense includes both of the
superoxide dismutase (SOD)-isoforms (Mn-SOD, Cu/Zn- injected into the left side.
SOD), which are induced to prevent oxidative and NO/ The 4 experimental groups groups (based on drug treatment)
-
ONOO -mediated damage [25]. were subdivided into 2 subgroups each. At 2 time points
Cell death and changes in neurite morphology were partly after the treatments, 3 h and 30 d, animals in the subgroups
reduced when the NO concentration was inhibited by NOS were decapitated. Heads were immediately frozen in liquid
o
inhibitors [23]. Our previous results indicated positive nitrogen and stored at -70 C until use. Crude mitochondrial
effects of pre-treatment with NOS inhibitors on the fraction preparations of forebrain cotices were used for the
development of neurotoxicity [33,38]. In view of the above biochemical analyses [11].
considerations, the present study was undertaken to examine
-
if the nitrite levels, ∙O2 production, MDA concentrations, Biochemical analyses
SOD activities and GSH contents that result after After deproteinization, the production of NO was evaluated
intracerebral injections of aluminium chloride (AlCl3) could by measuring nitrite and nitrate concentrations. Nitrites were
be modulated by pretreatment with N-nitro-L-arginine assayed directly by spectrophotometry at 492 nm using the
methyl ester (L-NAME), a non-specific NOS inhibitor. colorimetric method of Griess (Griess reagent: 1.5%
sulfanilamide in 1 M HCl plus 0.15% N-(1-naphthyl)
ethylendiamine dihydrochloride in distilled water). Nitrates
Materials and Methods
were first transformed into nitrites by cadmium reduction [24].
-
Superoxide anion (∙O2 ) content was determined by the
Animals
Male adult Wistar rats (500 ± 50 g) were used for these reduction of nitroblue-tetrazolium (Merck, Germany) in
- 20 Ivana D. Stevanović et al.
3 h (A) and 30 d (B) after the treatments. AlCl3 injection in the brain. Both Al and Abeta can potentate free radical
resulted in higher bilateral GSH concentrations after 3 h in formation by stabilizing iron in its more damaging ferrous
2+
the forebrain cortex that was significantly different compared (Fe ) form, which can promote the Fenton reaction. The
2+ 3+
to the controls (p ＜ 0.05). After 3 h, L-NAME + AlCl3 rate at which Fe was spontaneously oxidized to Fe was
injection resulted in lower GSH contents in both the ipsi- and significantly lower in the presence of Al salts [42]. In
-
contralateral forebrain cortices compared to the AlCl3- addition, neurotoxin injection resulted in increased ∙O2
treated group. Also, at 30 d after L-NAME + AlCl3 injection, production after 30 d in the ipsi- and contralateral forebrain
GSH contents were decreased in both the ipsi- and cortices compared to controls. Recent results indicate that
contralateral forebrain cortices compared to both the control microglia are CNS macrophages and are primary cellular
and the AlCl3-treated animals. After 3 h, L-NAME injection components of plaques that may contribute to the oxidative
resulted in lower bilateral GSH contents in the forebrain stress associated with chronic neurodegeneration [7].
2+
cortex compared to the AlCl3-treated group. However, after Al has been shown to alter Ca flux and homeostasis and
30 d, intrahippocampal L-NAME injection resulted in to facilitate the peroxidation of membrane lipids.
generally higher bilateral GSH contents in the forebrain Literature results suggest that Al may facilitate increases in
2+
cortex compared to the controls, as compared to the intracellular Ca and ROS, and potentially contribute to
AlCl3-treated animals and as compared to the L-NAME + neurotoxicity induced by other neurotoxicants [22]. We
AlCl3-treated animals (Fig. 5). have shown that at all test times (3 h, 30 d) post AlCl3
injection, there were increased bilateral MDA concentrations
in the forebrain cortex compared to the control animals.
Discussion
Furthermore, we have shown that AlCl3 injection resulted
The injection of AlCl3 into the CA1 sector of the rat in increased bilateral GSH concentrations after 3 h in the
hippocampus resulted in significant increases in nitrite forebrain cortex compared to controls. Literature results
- -
levels, ∙O2 production, MDA concentrations and GSH implicate that all detrimental effects of ONOO could be
contents in the forebrain cortex. successfully attenuated by the thiol-containing anti-oxidant
-
Numerous afferents from all areas of the cortex and the tripeptide glutathione [12]. Research shows that ONOO
thalamus represent the most important sourcees of can oxidatively modify both membranous and cytosolic
excitatory amino acids, whereas the nigrostriatal pathway proteins, which alters both their physical and chemical
and intrinsic circuits provide the striatum with dopamine, properties [15].
acetyl choline, GABA, NO and adenosine. Together, these It has been previously shown [37] that the interactions
neurotransmitter systems interact with each other and with between basalocortical and intracortical NOS neurons are
voltage-dependent conductances to efficiently regulate involved in the spatial and temporal regulation of cortical
synaptic transmission within this circuit [3]. perfusion following basal forebrain activation. Aluminium
In our study, AlCl3 injection resulted in increased nitrite accumulation in the brain can alter neuronal signal
concentrations after 3 h in the ipsilateral forebrain cortex, transduction pathways associated with glutamate receptors.
and bilateral nitrite concentrations were unchanged after Impairment of the Glu-NO-cGMP pathway in the brain
30 d in this brain structure compared to the controls. It has may be responsible for some of the neurological alterations
been previously shown [36] that production and oxidation that are induced by Al [4].
of NO in the brain increased in the early stages of disease, Decreased bilateral nitrite concentrations in the striatum at
while it decreased with increased loss of neurons. 30 d in the L-NAME + AlCl3 group, compared to the AlCl3-
The literature results implicate that the reaction product treated group, suggested that L-NAME suppressed nitrite
- -
between NO and ∙O2 , ONOO , is a strong oxidizing and production and decreased neuron impairment via N-methyl-
nitrating agent, which can react with all classes of D-aspartic acid (NMDA) receptors. Neuropharmacological
biomolecules. In the CNS, this product can be generated by data indicate that Abeta toxicity is mediated by an
microglial cells that are activated by pro-inflammatory excitotoxic cascade involving blockade of astroglial
-
cytokines or Abeta and by neurons when ONOO directly glutamate uptake, sustained activation of NMDA receptors
2+
contributes to the initiation of the neurodegenerative and an overt intracellular Ca influx. These changes are
process [40]. associated with increased NOS activity in cortical target
In our experiments, AlCl3 injection resulted in increased areas that may directly lead to the generation of free
-
∙O2 production after 3 h in the contralateral forebrain radicals [13]. A sustained overproduction of NO via NOS
cortex compared to controls. There are several lines of expression may be responsible, at least in part, for some of
evidence showing that Al is associated with oxidative the neurodegenerative changes caused by stress and
stress, possibly due to the pro-oxidant properties of Abeta support a possible neuroprotective role for NOS inhibitors
present in senile plaques. The presence of low molecular in this context [26].
-
weight Fe compounds can stimulate free radical production Decreased bilateral ∙O2 production in conjunction with
- The effect of L-NAME on AlCl3 toxicity in the rat brain 21
decreased MDA concentration, as well as decreased bilateral Greenwald RA (ed.). CRC Handbook of Methods for Oxygen
SOD activity, in the forebrain cortex 3 h and 30 d after Radical Research. pp. 123-132, CRC Press, Boca Raton,
1985.
L-NAME + AlCl3 injection, compared to AlCl3-treated
- 3. Calabresi P, Centonze D, Gubellini P, Marfia GA, Pisani
animals, suggested reduced enzyme substrate (∙O2 )
A, Sancesario G, Bernardi G. Synaptic transmission in the
production. In the same experimental group of animals, we
striatum: from plasticity to neurodegeneration. Prog Neurobiol
showed decreased GSH contents. Our results suggest the 2000, 61, 231-265.
importance of GSH participation in the glutathionylation Canales JJ, Corbalán R, Montoliu C, Llansola M, Monfort
4.
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against irreversible protein impairment [10]. impairs the glutamate-nitric oxide-cGMP pathway in cultured
Under our experimental conditions, 3 h after L-NAME neurons and in rat brain in vivo: molecular mechanisms and
injection, there was increased bilateral NO production in implications for neuropathology. J Inorg Biochem 2001, 87,
the forebrain cortex compared to the control group. 63-69.
Chabrier PE, Demerlé-Pallardy C, Auguet M. Nitric oxide
Literature results suggest that the inhibition of inducible 5.
synthases: targets for therapeutic strategies in neurological
NOS expression by L-NAME administration prevented an
diseases. Cell Mol Life Sci 1999, 55, 1029-1035.
increase in nitrogen intermediates and GABA release, but
6. Chakraborti A, Gulati K, Ray A. Age related differences in
not in glutamate release [41]. Also, recent results indicate
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NO donation by serving as an arginine analog [6]. In contrast, 194, 86-91.
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decreased NO concentrations, as well as decreased ∙O2 7. Colton CA, Chernyshev ON, Gilbert DL, Vitek MP.
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potential L-NAME anti-oxidative effects. Cucarella C, Montoliu C, Hermenegildo C, Sáez R, Manzo
8.
Decreased bilateral SOD activities along with decreased L, Miñana MD, Felipo V. Chronic exposure to aluminum
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J Neurochem 1998, 70, 1609-1614.
injection contributed to oxidative development at the early
9. Ferreira PC, Piai Kde A, Takayanagui AM, Segura-
time point. Thirty days post L-NAME injection showed
Muñoz SI. Aluminum as a risk factor for Alzheimer's
improvements of an oxidative stress development parameter
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- -
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